Myosin V is a two-headed unconventional myosin that has an extended neck due to the presence of six light chain binding IQ-repeats. In cells, myosin V is thought to be a vesicle motor. Double-headed and single-headed fragments of mouse myosin V, each tagged with the FLAG-epitope on their carboxyl-terminal end, were expressed in Sf9 cells along with calmodulin. In addition, we purified myosin V from mouse brain and chicken brain. Interestingly, the mouse brain myosin V contains only calmodulin for light chains, whereas the chicken brain myosin V contains bound essential light chain and calmodulin. The myosin V molecules have a high Vmax and a low KATPase of the steady state actin-activated MgATPase. There was no activation of the MgATPase activity of the recombinant fragments by calmodulin, but the activity of the intact myosin V molecules require calcium for maximal activation in solution. ADP markedly inhibited the actin-activated MgATPase activity and the in vitro motility. Several lines of evidence suggest that myosin V is a processive motor which can move some distance along actin filaments without dissociation. First, low concentrations of myosin V bound to the surface are able to translocate actin filaments. Second, the kinetics suggest that ADP release is partially or fully rate limiting and that the steady state intermediate is a strongly bound state. Third, single molecules are able to generate multiple steps in vitro using an optical trap. Fourth, electron microscopic data demonstrates that both heads of myosin V can bind to the same actin filament in the presence of ATP. The distance between the heads corresponds to 13 actin monomers (36 nm) or one helical repeat. Consistent with these data the optical trapping results show that the distance between stepping events in a run is 36 nm. In contrast, the first step in a run or the step size of a single attachment event is only 25 nm. This suggests that the second head must be able to search out the appropriate actin monomer while being tethered by the attached head. The power stroke takes place in two unequal steps.